Characteristics of human large granular lymphocytes and relationship to natural killer and K cells

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcγR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcγR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.     

Age-Related Differences in the Frequency of Ketoacidosis at Diagnosis of Type 1 Diabetes in Children and Adolescents

OBJECTIVE

We studied the prevalence of diabetic ketoacidosis (DKA) at diagnosis of type 1 diabetes in children in Finland.

RESEARCH DESIGN AND METHODS

From 2002 to 2005, data on virtually all children <15 years of age diagnosed with type 1 diabetes (n = 1,656) in Finland were collected.

RESULTS

DKA was present in 19.4% of the case subjects, and 4.3% had severe DKA. In children aged 0–4, 5–9, and 10–14 years, DKA was present in 16.5, 14.8, and 26.4%, respectively (P < 0.001). Severe DKA occurred in 3.7, 3.1, and 5.9%, respectively (P = 0.048). DKA was present in 30.1% and severe DKA in 7.8% of children aged <2 years.

CONCLUSION

The overall frequency of DKA in children is low in Finland at diagnosis of type 1 diabetes. However, both children <2 years of age and adolescents aged 10–14 years are at increased risk of DKA.The incidence of diabetic ketoacidosis (DKA) in children with newly diagnosed type 1 diabetes may be decreasing in developed countries (1,2).     

Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk

Aims

To determine the relative frequency of mutations in three different genes (low‐density lipoprotein receptor (LDLR), APOB, PCSK9), and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK.

Patients and methods

409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single‐strand conformational polymorphism (SSCP) (exons 3, 4, 6–10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP.

Results

Mutations were detected in 253 (61.9%) patients: 236 (57.7%) carried LDLR, 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels (LDLR, PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post‐statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL‐C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL‐C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03).

Conclusions

The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.Familial hypercholesterolaemia is an autosomal dominant disorder associated with increased risk of coronary heart disease (CHD), with an estimated prevalence in the UK of 1 in 500 to 1 in 600.¹ Roughly half of the men with familial hypercholesterolaemia, if untreated, will have developed clinically evident CHD by the age of 55 years. Affected women from the same families typically develop CHD about 9 years later than their affected male relatives, but again, often remarkably prematurely.² The proportion of patients with familial hypercholesterolaemia identified and being treated in lipid clinics to date in the UK is, at best, 15% of the predicted number, with most of these being young people.¹ Because lipid‐lowering drug treatment with statins substantially reduces coronary morbidity and mortality,³ identification of affected people by screening is crucially important. To this end, the Department of Health has recently funded five pilot sites in the UK to determine the efficiency of cascade testing in the current social structure and the framework of the National Health Service. Cascade testing is a cost‐effective method of finding additional patients with familial hypercholesterolaemia,⁴ and has been used extensively in other countries in Europe, most notably in Holland,⁵ for the past 5 years.

Key points

• Patients with familial hypercholesterolaemia with a detectable LDLR mutation have a higher risk of early CHD than those in whom no mutation was detected.
• Patients with the pD374Y mutation in PCSK9 have the highest pretreatment and post‐treatment levels of plasma cholesterol and the highest risk of early CHD.
• Mutations in PCSK9 appear to be uncommon in patients with familial hypercholesterolaemia in UK.

The extent to which DNA testing for familial hypercholesterolaemia complements cholesterol measurement in cascade screening to identify affected patients is unclear, as is its role in determining the risk of CHD and response to treatment. In the current study, we carried out molecular genetic testing in patients recruited from the Simon Broome familial hypercholesterolaemia register⁶ in the UK as a cross‐sectional cohort study to identify risk factors for premature CHD in patients with familial hypercholesterolaemia.⁷ Primary results from this study indicated that the conventional cardiovascular risk factors of age, sex, smoking, pretreatment cholesterol levels and low levels of high‐density lipids (especially in women) were all associated with higher risk of CHD,⁷ confirming associations reported in other studies, for example.⁸ When this UK study was started, mutations at two loci causing familial hypercholesterolaemia had been identified, with mutations in the low‐density lipoprotein receptor gene (LDLR) accounting for most of the identified mutations,⁹ whereas one particular mutation in the gene encoding the ligand for the low density lipoprotein (LDL) receptor—namely apolipoprotein B (FDB)—occurs in about 5% of patients in the UK.¹⁰ This mutation, which alters a single amino acid (p.R3500Q), has been shown to reduce the affinity of the LDL cholesterol particle,¹⁰ where ApoB is the single‐protein component for the receptor. For the LDLR, currently >100 mutations have been reported in UK patients to date^9,11 (see also www.ucl.ac.uk/FH). A commercially available kit for screening for deletions and rearrangements of the LDLR gene has become available, and it is known that up to 5% of patients with familial hypercholesterolaemia in patients in the UK may have such a deletion.¹²Recently, defects in a third gene causing monogenic hypercholesterolaemia have been identified.¹³ The gene protein convertase subtilisin/kexin type 9 (PCSK9) codes for an enzyme that has also been called “neural apoptosis regulated convertase 1”, which has recently been proposed to be involved in degrading the LDLR protein in the lysosome of the cell and preventing it from recycling.¹⁴ Gain‐ of‐ function mutations in the PCSK9 gene could therefore cause increased degradation of LDLRs, reduced numbers of receptors on the surface of the cell, and monogenic hypercholesterolaemia. An alternative mechanism has also been proposed for the hypercholesterolaemic effect, whereby the gain of function causes increased secretion of apoB‐containing lipoproteins from the liver, with this being supported by in vivo turnover studies in patients carrying PCSK9 missense mutations¹⁵ and by in vitro studies in transiently transfected rat liver cells.¹⁶ One mutation in this gene, p.D374Y, has been reported in several independent families^16,17,18 and we therefore also tested for this cause of familial hypercholesterolaemia in this group of patients, as well as using single‐strand conformational polymorphism (SSCP) analysis and direct sequencing to screen all coding exons of the gene.Although patients with no identified mutation may have a monogenic cause of the disorder in an as yet undiscovered gene, it is also possible that some may have polygenic hypercholesterolaemia and have been misclassified using current clinical diagnostic criteria. These patients would be expected to have a milder degree of hyperlipidaemia, possibly not present from birth but only developing in later life, and would therefore be predicted to have a lower risk of CHD. The hypothesis we set out to test was that patients with identified mutations in the LDLR, PCSK9 or APOB genes would be at greater risk of CHD than patients with no identified mutation.     

Effects of tibolone and hormone replacement therapy on the breast of cynomolgus monkeys

OBJECTIVE: To measure the effects of 2 years of treatment with tibolone on the breasts of cynomolgus macaques (Macaca fascicularis) in comparison with conventional hormone replacement therapy. DESIGN: Ovariectomized cynomolgus macaques were randomized into five groups and treated for 2 years. Groups included controls (n = 31) and four drug treatments, including tibolone at 0.05 mg/kg (LoTIB, n = 30) or 0.2 mg/kg (HiTIB, n = 31), conjugated equine estrogens at 0.042 mg/kg (CEE, n = 28), or CEE + medroxyprogesterone acetate (MPA) at 0.167 mg/kg (CEE + MPA, n = 29). Endpoints included histologic, histomorphometric, and immunohistochemical assessment of the mammary gland. RESULTS: Tibolone did not cause stimulation of the breast in contrast to distinct proliferative responses of the breast to CEE and CEE + MPA, as measured by increases in breast epithelial tissue area and expression of the proliferation marker Ki67 in breast epithelial cells. Tibolone at the higher dose increased progesterone receptor expression in the breast relative to controls, indicating partial estrogen-agonist activity, but without induction of proliferation. Progesterone receptor expression was also induced by CEE. CONCLUSIONS: Tibolone may have an advantage over conventional hormone replacement therapy because it does not stimulate proliferation in the breast. This lack of mammotrophic effect may reflect a lower risk for promotion of breast cancer.     

Ovarian stimulation with pure follicle-stimulating hormone/human menopausal gonadotropin and improved laparoscopic aspiration needles influence the success of an in vitro fertilization program

The effect of a combined pure follicle-stimulating hormone/human menopausal gonadotropin (pFSH/hMG) ovarian stimulation regimen and modified, sharpened laparoscopic follicular aspiration needles on the number of oocytes retrieved and the oocyte/follicle ratio in 43 consecutive cycles of in vitro fertilization (IVF) were retrospectively compared with 99 consecutive preceeding cycles stimulated with hMG alone and captured with aspiration needles that had never been sharpened. A modified laparoscopic follicular aspiration needle is described. Purified FSH/hMG ovarian stimulation significantly improved the mean serum estradiol levels, number of preovulatory follicles, and, therefore, the total number of oocytes recovered per cycle. The mean ratios of oocytes recovered per preovulatory follicle documented on ultrasound, and per aspirated follicle, increased significantly using sharpened needles. Both modifications improved the success rate of our IVF program.     

Cerebral palsy rates among low-birthweight infants fell in the 1990s

Using a population-based register, this study sought to ascertain changes in the rate and severity of cerebral palsy (CP) in a geographically defined area of the UK among infants weighing less than 1500 g and born between 1984 and 1995. There were 417414 live births in the area, which included Berkshire, Buckinghamshire, Northamptonshire, and Oxfordshire. Of the 898 children with CP (526 males, 372 females), 194 (21.6%) weighed less than 1500 g at birth. The overall CP rate for neonatal survivors fell from 2.5 out of every 1000 in 1984 to 1986 to 1.7 in 1993 to 1995. The rate for those weighing less than 1000 g rose to 90 out of every 1000 neonatal survivors in 1987 to 1989 and then fell to 57 in 1993 to 1995. A similar pattern is seen among infants weighing 1000 to 1499 g at birth, the rate rising to 77 in 1987 to 1988 and then falling to 40 in 1993 to 1995. The rate of severe motor disability among infants weighing less than 1500 g also decreased (24.6 in 1984-1986 to 12.5 in 1993-1995). The relation of these findings to changes in perinatal care in the early 1990s is not known.     

Increased level of polymerase Ⅲ transcribed Alu RNA in hepatocellular carcinoma tissue

There have been extensive observations that RNA containing repetitive elements accumulates in transformed cells and tumor tissues. In the present study, we first obtained result consistent with previous observations by in situ hybridization.     

Seizures in children with low-grade tumors： outcome after tumor resection and risk factors for uncontrolled seizures

OBJECT： The goals of this study were to define the incidence of seizures in children with low-grade tumors, study seizure outcome after lesionectomy in these children, and identify risk factors for poor seizure outcome, METHODS： The authors performed a retrospective chart review of children who harbored low-grade brain tumors, experienced seizures, and were treated in a single institution, Statistical analyses included step-wise as well as single-variable binary logistic regression analyses.